In grain crops of agronomic importance, seed formation is the ultimate goal of plant development. Seeds are harvested for use in food, feed, and industrial products. The utility and value of those seeds are determined by the quantity and quality of protein, oil, and starch contained therein. In turn, the quality and quantity of seed produced may be affected by environmental conditions at any point prior to fertilization through seed maturation. In particular, stress at or around the time of fertilization may have substantial impact on seed development. Members of the grass family (Poaceae), which include the cereal grains, produce dry, one-seeded fruits. This type of fruit is, strictly speaking, a caryopsis but is commonly called a kernel or grain. The caryopsis of a fruit coat or pericarp surrounds the seed and adheres tightly to a seed coat. The seed consists of an embryo or germ and an endosperm enclosed by a nucellar epidermis and a seed coat. Accordingly the grain comprises the seed and its coat or pericarp. The seed comprises the embryo and the endosperm. (R. Carl Hoseney in “Principles of Cereal Science and Technology” expressly incorporated by reference in its entirety).
A fertile corn plant contains both male and female reproductive tissues, commonly known as the tassel and the ear, respectively. The tassel tissues form the haploid pollen grains with two nuclei in each grain, which, when shed at anthesis, contact the silks of a female ear. The ear may be on the same plant as that which shed the pollen, or on a different plant. The pollen cell develops a structure known as a pollen tube, which extends down through an individual female silk to the ovule. The two male nuclei travel through this tube to reach the haploid female egg at the base of the silk. One of the male nuclei fuses with and fertilizes the female haploid egg nuclei to form the zygote, which is diploid in chromosome number and will become the embryo within the kernel. The remaining male nucleus fuses with and fertilizes a second female nucleus to form the primary endosperm nucleus, which is triploid in number and will become the endosperm of the kernel, or seed, of the corn plant. Non-fertilized ovules do not produce kernels and the unfertilized tissues eventually degenerate.
The kernel consists of a number of parts, some derived from maternal tissue and others from the fertilization process. Maternally, the kernel inherits a number of tissues, including a protective, surrounding pericarp and a pedicel. The pedicel is a short stalk-like tissue which attaches the kernel to the cob and provides nutrient transfer from maternal tissue into the kernel. The kernel contains tissues resulting from the fertilization activities, including the new embryo as well as the endosperm. The embryo is the miniature progenitor of the next generation, containing cells for root and shoot growth of a new, young corn plant. It is also one tissue in which oils and proteins are stored in the kernel. The endosperm functions more as a nutritive tissue and provides the energy in the form of stored starch, proteins and oil, needed for the germination and initial growth of the embryo.
Considering the complex regulation that occurs during embryo and kernel development in higher plants, and considering that it is commonly grain that is a primary source of nutrition for animals and humans, key tools needed to improve such a nutritional source include genetic promoters that can drive the expression of nutrition enhancing genes. On the other hand the embryo is highly sensitive toward stresses. Stresses to plants may be caused by both biotic and abiotic agents. For example, biotic causes of stress include infection with a pathogen, insect feeding, and parasitism by another plant such as mistletoe, and grazing by ruminant animals. Abiotic stresses include, for example, excessive or insufficient available water, insufficient light, temperature extremes, synthetic chemicals such as herbicides, excessive wind, extremes of soil pH, limited nutrient availability, and air pollution. Yet plants survive and often flourish, even under unfavorable conditions, using a variety of internal and external mechanisms for avoiding or tolerating stress. Plants' physiological responses to stress reflect changes in gene expression.
While manipulation of stress-induced genes may play an important role in improving plant tolerance to stresses, it has been shown that constitutive expression of stress-inducible genes has a severe negative impact on plant growth and development when the stress is not present. (Kasuga 1999) Therefore, there is a need in the art for promoters driving expression which is temporally- and/or spatially-differentiated, to provide a means to control and direct gene expression in specific cells or tissues at critical times, especially to provide stress tolerance or avoidance. In particular, drought and/or density stress of maize often results in reduced yield, typically from plant failure to set and fill seed in the apical portion of the ear, a condition known as “tip kernel abortion” or colloquially as “nosing back.” To stabilize plant development and grain yield under unfavorable environments, manipulation of hormones and carbon supply to the developing ear and its kernels is of interest. Thus there is a need for promoters which drive gene expression in female reproductive tissues under abiotic stress conditions.
One other well-known problem in the art of plant biotechnology is marker-deletion. Selectable marker are useful during the transformation process to select for, and identify, transformed organisms, but typically provide no useful function once the transformed organism has been identified and contributes substantially to the lack of acceptance of these “gene food” products among consumers (Kuiper 2001), and few markers are available that are not based on these mechanisms (Hare 2002). Thus, there are multiple attempts to develop techniques by means of which marker DNA can be excised from plant genome (Ow 1995; Gleave 1999). The person skilled in the art is familiar with a variety of systems for the site-directed removal of recombinantly introduced nucleic acid sequences. They are mainly based on the use of sequence specific recombinases. Various sequence-specific recombination systems are described, such as the Cre/lox system of the bacteriophage P1 (Dale 1991; Russell 1992; Osborne 1995), the yeast FLP/FRT system (Kilby 1995; Lyznik 1996), the Mu phage Gin recombinase, the E. coli Pin recombinase, the R/RS system of the plasmid pSR1 (Onouchi 1995; Sugita2000), the attP/bacteriophage Lambda system (Zubko 2000). It is one known disadvantage of these methods known in the prior art that excision is not homogenous through the entire plants thereby leading to mosaic-like excision patterns, which require laborious additional rounds of selection and regeneration.
Promoters that confer enhanced expression during seed or grain maturation are also described (such as the barley hordein promoters; see US patent application 20040088754). Promoters which direct embryo-specific or seed-specific expression in dicots (e.g., the soybean conglycinin promoter; Chen 1988; the napin promoter, Kridl 1991) are in general not capable to direct similar expression in monocots. Unfortunately, relatively few promoters specifically directing this aspect of physiology have been identified (see for example US20040163144).
The octopine synthase (ocs) and mannopine synthase (mas) gene promoters have been used to direct the expression of linked genes in transgenic plants. However, the application of these promoters has been restricted by weak expression levels in certain tissues of transgenic plants (DiRita 1987; Harpster 1988; Sanger 1990). For example, the ocs promoter directs a distinct cell-specific pattern of expression in transgenic tobacco (Kononowicz 1992). The mas gene exhibits weak expression in leaves and stems, but has stronger expression in roots and exhibits a degree of wound and auxin inducibility (Langridge 1989; Teeri 1989; Saito 1991; Guevara-Garcia 1993). Chimeric promoters for expressing genes in plants comprising Agrobacterium tumefaciens opine synthase upstream activating sequences operably linked to a Agrobacterium tumefaciens opine synthase promoter are described (Ni 1995; U.S. Pat. No. 5,955,646). The most characterized sequence is the so called “super-promoter”, a chimeric construct of three upstream activating sequences derived from an Agrobacterium tumefaciens octopine synthase gene operably linked to a transcription regulating nucleotide sequence derived from the promoter of an Agrobacterium tumefaciens mannopine synthase gene. Although the promoter is widely used in dicotyledonous plants, its experiences from application to monocotyledonous plants are very limited. Kononov et al. (A Comparative Study of the Activity of the Super-promoter with Other Promoters in Maize (1999) 20th annual crown gall conference, University of Texas-Houston Medical School; abstract book, p. 36; Comparative Study of the Activity of the Super-promoter and Other Promoters in Maize (1998) 19th annual crown gall meeting, Purdue University, West Lafayette, Ind.]) showed expression a broad range of tissues. Expression was nearly the same in all tissues but was elevated in roots. In contrast to the ubiquitin promoter the presence or absence of an intron sequence was reported to have no effect on transcription activity of the super-promoter.
Accordingly there is a first need in the art for promoter sequences which allow for expression in starch endosperm during seed development and in embryo during the early germinating seed. Further more there is a strong second need in the art for promoter sequences which allow for strong expression of excision mediating enzymes in a way that the resulting plant is substantially marker-free.
For the first need in the art some seed- or grain-specific promoters are described include those associated with genes that encode plant seed storage proteins such as genes encoding: barley hordeins, rice glutelins, oryzins, prolamines, or globulins; wheat gliadins or glutenins; maize zeins or glutelins; oat glutelins; sorghum kafirins; millet pennisetins; or rye secalins. However, on the one hand expression of these promoters is often leaky or of low expression level. Furthermore, it has been noted that improvement of crop plants with multiple transgenes (“stacking”) is of increasing interest. For example, a single maize hybrid may comprise recombinant DNA constructs conferring not only insect resistance, but also resistance to a specific herbicide. Importantly, appropriate regulatory sequences are needed to drive the desired expression of each of these or other transgenes of interest. Furthermore, it is important that regulatory elements be distinct from each other. Concerns associated with the utilization of similar regulatory sequences to drive expression of multiple genes include, but are not restricted to: (a) pairing along homologous regions, crossing-over and loss of the intervening region either within a plasmid prior to integration, or within the plant genome, post-integration; (b) hairpin loops caused by two copies of the sequence in opposite orientation adjacent to each other, again with possibilities of excision and loss of these regulatory regions; (c) competition among different copies of the same promoter region for binding of promoter-specific transcription factors or other regulatory DNA-binding proteins.
There is, therefore, a great need in the art for the identification of novel sequences that can be used for expression of selected transgenes in economically important plants, especially in monocotyledonous plants. It is thus an objective of the present invention to provide new and alternative expression cassettes for endosperm- and/or embryo-preferential or specific expression. The objective is solved by the present invention.